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Old 04-19-2017, 10:58 AM   #1
icanwinwyz
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Location: LA

Join Date: Feb 2016
Posts: 8
Default RNA-seq alignment on novel region

Hi everyone,
My colleagues just discovered a novel lncRNA region and I would like to check its expression in my 30 RNA-seq samples. The length of this region is 2438bp with two transcript variants, which are 645bp and 553bp (strand information is not available), respectively. I used STAR to perform the alignment on this region and bedtools multicov to count the expression on whole region and its two transcript variants. The procedure is shown below:

STAR --runMode genomeGenerate --genomeDir ./lncRNA-genome --genomeFastaFiles ./lncRNA.fa --genomeSAindexNbases 10 ####I extracted the sequences of this region and made fasta file and built up STAR index, since the length is small, so I have to change the "genomeSAindexNbases"

STAR --genomeDir ./lncRNA_genome --outSAMunmapped Within --runThreadN 4 --genomeLoad LoadAndRemove --limitBAMsortRAM 10000000002 --outSAMtype BAM SortedByCoordinate --outFileNamePrefix sample1 --readFilesIn sample1.fastq ######STAR alignment only to this region

samtools index sample1.sorted.bam####index bam

bedtools multicov -bams sample1.sorted.bam -bed ./lncRNA.bed > results.txt#####bedtools to count expression on whole region and two variants

After counting the expression, I would like to use rpm (reads per million) to compare the expression for the whole region and two transcript variants among my 30 RNA-seq samples, respectively.

So I would like to know is there anything that I miss or have to pay attention to or you guys have some suggestions? Thanks!!
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